Array for crystallizing protein, device for crystallizing protein and method of screening protein crystallization using the same

ABSTRACT

The present invention relates to a method of precipitating protein crystals from a protein-containing sample. The present invention also relates to a novel microarray and a novel device for screening for protein crystallization condition. Furthermore, the present invention relates to a method of conveniently and quickly screening for protein crystallization conditions using the microarray or the device having highly integrated and held crystallization conditions even with an extremely small quantity of a sample.

TECHNICAL FIELD

The present invention relates to a method of precipitating proteincrystals from a protein-containing sample. The present invention relatesto a novel microarray and a novel device for screening for proteincrystallization condition. Furthermore, the present invention relates toa method of conveniently and quickly screening for proteincrystallization conditions from an extremely small quantity of a sampleusing the microarray or the device having highly integratedcrystallization conditions.

BACKGROUND ART

In recent years, the so-called structural genome science movement hasbeen expanding. This involves the exhaustive analysis of proteinstructure, based on which mechanisms of living phenomena areinvestigated. In the field of the structural genome science, furtherlabor saving and increased speed are required for the process ofstructural analysis. To meet these needs, the development of a method ofscreening for crystallization conditions quickly and highly efficientlyusing a minute quantity of a protein sample is expected.

Analysis of a three-dimensional protein structure requires good crystalsthereof. Thus, that a large quantity of samples are used and a long timeis spent in searching for conditions for preparing such crystals, whichis a current problem. That is, existing methods require very complicatedprocedures and thus have been inefficient, in addition to requiring alarge quantity of protein samples.

For this purpose, reagent kits for screening for crystallization havebeen developed, and circumstances relating to the time for preparingscreening reagents required for screening for crystallization conditionshave become improved. For example, JP Patent Publication (Kokai) No.9-89898 proposes an immobilized reagent for screening for proteincrystallization conditions, which comprises a solid support having aplural number of types of protein-precipitating agents immobilizedthereon. However, including this case, conventional screening forcrystallization conditions is conducted using a solution volume ofseveral microliters per condition. The search time for crystallizationconditions reaches several hours to few days. Therefore, a furthersmaller quantity of a sample and a shorter search time forcrystallization conditions are expected.

In the meantime, with the above background, various techniques have beendeveloped as methods of crystallizing protein. The major techniques ofthese methods are the batch method, the vapor diffusion method, and theliquid-liquid diffusion method (or dialysis method).

The “batch method” involves adding the solution of a precipitating agentto a protein solution in driblets so as to make the solution slightlymuddy, removing insoluble matters by centrifugation, putting thesupernatant in a small test tube or the like, sealing the tube, and thenallowing the resultant to stand. Although the operation of this methodis convenient, protein crystals are unable to be efficiently obtainedthereby. This is because crystals cannot be easily obtained, the qualityof the resulting crystals is poor (minute crystals are precipitated),and the active control of crystallization conditions is required.

The “vapor diffusion method” involves placing a droplet of a proteinsolution containing a precipitating agent in a container containing abuffer (external solution) containing a precipitating agent with ahigher concentration, sealing the chamber, and then allowing it tostand. Depending on how the droplet is placed, the hanging drop methodand the sitting drop method may be used. The hanging drop methodinvolves spotting a small droplet of a protein solution onto a coverglass and inverting the cover glass over a reservoir so as to seal thereservoir. The sitting drop method involves placing an appropriatedroplet stand within a reservoir, spotting a small drop of a proteinsolution on the droplet stand, and then sealing the reservoir using acover glass or the like. The vapor diffusion method is thought to beappropriate for screening for wide-ranging crystallization conditions,because the concentration of a protein solution and that of aprecipitating agent change over time, and only a small quantity ofprotein is required for use. A screening method of proteincrystallization conditions utilizing the vapor diffusion method has beenreported (see the WO 00/60345 pamphlet).

Such a screening method requires an expensive large-scale systeminvolving high throughput in order to examine a large number ofconditions using a more minute quantity of a sample.

The “liquid-liquid diffusion method,” or “dialysis method,” involvesbringing a protein solution into contact with a buffer (externalsolution) containing a precipitating agent via the interface or gel(liquid-liquid diffusion method), or a semipermeable membrane (dialysismethod) as a boundary between the solutions, thereby graduallyincreasing the concentration of the precipitating agent in the proteinsolution. These methods have the advantages of both the above-describedvapor diffusion method and batch method, so that these methods areappropriate for obtaining protein crystals with good quality. However,in these methods, a relatively large volume of protein solution is used,and it is also difficult to make the volume thereof less. Moreover, theprocedure therefor is complicated and requires much time, so that itcannot be said to be convenient.

Other than the above-described methods, various techniques have beendeveloped. For example, a technique for crystallizing protein, whichutilizes rotation (in other words, gravity) to obtain protein crystalswith good quality, has been reported (e.g., JP Patent Publication(Kokai) No. 2002-316899). However, this technique requires a rotationapparatus, so that the whole system becomes excessively large. Inaddition, since the protein quantity required for this system isapproximately 20 μL, the system is inappropriate for a case where only asmall quantity of protein is obtained. Furthermore, for example, as animproved vapor diffusion method, a system for crystallizing proteinutilizing potential has been reported (e.g., JP Patent Publication(Kokai) No. 2001-213699 and JP Patent. Publication (Kokai) No.2002-179500). With this system, crystals are grown by adsorbing proteinmolecules on solids utilizing differences in the surface potentials ofthe solids, and crystals can be grown even when a small quantity of aprotein solution is used. However, the system requires the changing ofchips (solids) depending on types of protein to be crystallized.

Moreover, a method whereby crystallization is performed in a containerprovided with a sandwich structure comprising gel containing the proteinto be crystallized and gel containing a precipitating agent has beenreported (JP Patent Publication (Kokai) No. 6-321700). This techniqueinvolves bringing the gel containing the protein into contact with thegel containing the precipitating agent, turning them around so as todiffuse the protein and the precipitating agent between both gels, andcausing the protein to react with the agent, thereby crystallizing theprotein. However, with this technique, only a reaction condition between1 type of protein and 1 type of a precipitating agent (concentration andtype) can be examined per operation, and thus the method cannot be saidto be efficient.

For the reasons described above, a technique to conveniently, highlyefficiently, and economically obtain protein crystals in a good statefor X-ray diffraction or the like has been desired.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method ofconveniently and highly efficiently crystallizing protein to obtain goodcrystals, and a device and a method therefor that enables screening forprotein crystallization condition within a short time using a minutequantity of a sample.

As a result of intensive studies to achieve the above objects, we havefound that the crystallization conditions of a specific protein can beconveniently and quickly determined using a microarray comprising aplural number of types and concentrations of protein crystallizationagents held therein, and using a minute quantity of a sample.Furthermore, we have found that good crystals can be grown by applying aprotein-containing sample to gel holding protein crystallization agents,and then bringing the crystallization agents into contact with theprotein. Thus, we have completed the present invention.

Hence, the present invention relates to a method of crystallizingprotein, which comprises the following steps of:

-   -   (a) applying a protein-containing sample to polymer gel holding        protein crystallization agents; and    -   (b) bringing the above protein into contact with the above        protein crystallization agents.

In the method of crystallizing protein, the protein-containing samplemay contain a protein solubilizing agent and/or an additive, in additionto the protein to be crystallized. Moreover, the polymer gel holding theprotein crystallization agents is preferably transparent.

Examples of the protein crystallization agent include a precipitatingagent, a pH buffering agent, and any combination thereof. Examples ofthe precipitating agent include sodium chloride, polyethylene glycol,2-methyl 2,4-pentanediol, and ammonium sulfate. Examples of the pHbuffering agent include sodium acetate trihydrate, potassium phosphate,imidazole, sodium citrate, and sodium cacodylate.

Furthermore, the present invention relates to a microarray for proteincrystallization, which comprises at least 10 different types and/ordifferent concentrations of protein crystallization agents held thereinper 1 cm² of the microarray surface.

In the microarray for protein crystallization, the proteincrystallization agents are preferably held by means of the polymer gel.In addition, the polymer gel holding the protein crystallization agentsis preferably transparent.

In addition, the microarray for protein crystallization may comprise aplurality of hollow fibers arrayed therein holding different typesand/or different concentrations of the protein crystallization agents inthe hollow portions thereof. In this case, the protein crystallizationagents can be held by means of the polymer gel that fills the hollowportions.

Examples of the above protein crystallization agent include aprecipitating agent, a pH buffering agent, and any combination thereof.Here, examples of the precipitating agent include sodium chloride,polyethylene glycol, and ammonium sulfate. Examples of the pH bufferingagent include sodium acetate trihydrate, potassium phosphate, imidazole,sodium citrate, and sodium cacodylate.

Furthermore, the present invention relates to a device for proteincrystallization, which is provided with the following (a) and (b):

-   -   (a) a microarray for protein crystallization having        protein-crystallization-agent-holding portions that comprise at        least 10 different types and/or different concentrations of        protein crystallization agents held thereon per 1 cm² of the        microarray surface; and    -   (b) a plate having wells that can be filled with a        protein-containing sample, and correspond to each        protein-crystallization-agent-holding portion of the        microarray (a) above for protein crystallization.

In the above (a), the protein crystallization agent is held by means ofthe polymer gel.

In (a), examples of the protein crystallization agent include aprecipitating agent, a pH buffering agent, and any combination of these.In addition in (a), the microarray for protein crystallization maycomprise a plurality of hollow fibers arrayed therein holding differenttypes and/or different concentrations of the protein crystallizationagents in the hollow portions thereof. In this case, the proteincrystallization agents can be held by means of the polymer gel fillingthe hollow portions.

Furthermore in (b), the capacity of each well is preferably 1 μL orless. Furthermore in (b), when the plate is superposed on the microarray(a) for protein crystallization, the superposed portion corresponding toeach protein-crystallization-agent-holding portion of the array ispreferably transparent. Moreover, the plate is preferably made of anoptically transparent material, such as a transparent resin or glass.

In (b) above, the plate can have a structure wherein a plate materialperforated regularly to have a plurality of holes is adhered to asubstrate. Here, the substrate is preferably prepared from an opticallytransparent material, such as a slide glass. Moreover, the platematerial preferably is made of material selected from the groupconsisting of metal, resin and rubber.

The microarray (a) above for protein crystallization may be fixed on asupport. Here, the support is preferably made of an opticallytransparent material such as a slide glass. In addition, marking can bemade on the support, so as to determine a position to which themicroarray (a) for protein crystallization is fixed.

Furthermore, in the device for protein crystallization, a spacer can beplaced between the support and the plate (b), where the spacer has spaceinto which the microarray (a) for protein crystallization fixed on thesupport can be accommodated. Here, the spacer is made of materialselected from the group consisting of metal, resin, and rubber.

Moreover, in the above microarray for protein crystallization or theabove device for protein crystallization, the present invention relatesto a method of screening for protein crystallization condition, whichcomprises a step of precipitating protein by bringing the proteincrystallization agents into contact with a protein-containing sample onand/or in the microarray. Here, the protein-containing sample mayfurther contain a protein solubilizing agent and a protein stabilizingagent such as a reducing agent, in addition to protein to be screenedfor.

In addition, the above screening method can be conducted under aplurality of temperature conditions.

Furthermore, the volume of the protein-containing sample solution usedin the above screening method is preferably 1 μL or less per type orconcentration of the protein crystallization agent.

Furthermore, in the above device for protein crystallization, thepresent invention relates to a method of screening for proteincrystallization conditions, which comprises a step of precipitatingprotein by superposing the wells of (b) filled with theprotein-containing sample onto the protein-crystallization-agent-holdingportions holding the protein crystallization agents on the microarray(a), so as to bring the protein crystallization agents into contact withthe protein.

The terms relating to the present invention are as defined below.

The term “protein” used in this specification includes natural orsynthetic peptides, polypeptides, protein, and protein complexes. Thesesubstances can be purified by extraction and isolation from natural orsynthetic materials, or generation by genetic engineering techniques,chemical synthesis techniques, or the like, and then a combined use ofgeneral purification methods such as solvent extraction, columnchromatography, liquid chromatography, and the like.

The term “crystallization” used in this specification refers toobtaining crystals by growing or precipitating crystals from a proteinsolution.

The term “protein-containing sample” used in this specification refersto a sample containing protein to be crystallized or protein whosecrystallization conditions are to be specified.

The term “protein crystallization agent” used in this specificationmeans a compound that acts to lower the solubility of protein. Examplesof the protein crystallization agent include a precipitating agent, a pHbuffering agent, other additives.

The term “hold” used in this specification means to immobilize theprotein crystallization agent to polymer gel or a microarray.

The term “polymer” used in this specification includes any naturalpolymers (e.g., biopolymers) and synthetic polymers.

The term “polymer gel” used in this specification means polymer gelprepared by polymerizing or gelatinizing a polymerizable monomer or asolution thereof by a polymerization reaction, or polymer gel preparedby causing a solution to lose its flow property by adding a syntheticpolymer or a natural polymer.

The term “apply” used in this specification refers to, for example,adding a protein-containing sample dropwise toprotein-crystallization-agent-holding portions, filling the portionswith the sample manually or mechanically using a syringe or the like, orimmersing a microarray in a protein-containing sample.

The term “bring . . . into contact with” used in this specificationrefers to, for example, bringing a protein-containing sample near to aprotein crystallization agent to a degree such that a reaction can occurbetween the two.

The term “microarray” used in this specification refers to, generally, amicroarray having a configuration wherein many substances for reactionare arranged in order to conduct many reactions on a minute support.

The term “hollow portion” used in this specification means, for example,a hollow portion within a tubular or a hollow fiber.

The term “hollow fiber” used in this specification means a tubular or astraw-shaped fiber whose inside is hollow.

The term “protein-crystallization-agent-holding portion” used in thisspecification refers to a portion holding a protein crystallizationagent. Examples of such a portion in the present invention includepolymer gel or array portions of a microarray.

The term “fill” used in this specification means to fill a portion byputting a protein-containing sample into the portion.

The term “well” used in this specification means a part that is dentedcompared with the surroundings. Examples of such a well include thosehaving various shapes such as holes and grooves.

The term “optically transparent material” used in this specificationmeans a material that is transparent and through which light istransmittable.

The term “(to create a) hole” used in this specification means to createholes and the resulting holes.

The term “regularly” used in this specification refers to array in anorderly manner holes within a frame with a certain size so that thenumber of fibers or the number of holes become fixed.

The term “plate material” used in this specification means a member forpreparing a plate, which is made of material and has a shape that enablethe formation of wells that can be filled with a protein-containingsample.

The term “substrate” used in this specification means a member to whicha plate material is adhered in order to prepare a plate.

The term “adhere” used in this specification means to bring and keepmembers into contact with each other.

The term “support” used in this specification means a member to which amicroarray for protein crystallization is fixed.

The term “fix” used in this specification means to secure an object to aposition.

The term “marking” used in this specification means to make a mark thatis an indication (label).

The term “can be accommodated” used in this specification means that amember can be placed into another member or between members.

The term “space” used in this specification means an opening or a holeof a member.

The term “spacer” used in this specification means a member to be usedto provide a distance between members.

The term “screening” used in this specification means the step ofselecting conditions for protein to crystallize, and the step ofnarrowing appropriate conditions from among many conditions.

The present invention is explained in detail as follows. Thisapplication claims a priority from JP Patent Application No. 2001-376972filed on Dec. 11, 2001, and JP patent application No. 2002-229173 filedon Aug. 6, 2002, and includes part or all of the contents as disclosedin the specifications and drawings of the above patent applications.

The present invention relates to a method of crystallizing protein,which comprises applying a protein-containing sample to polymer gelholding protein crystallization agents, and bringing the protein intocontact with the protein crystallization agents utilizing the diffusionphenomenon of the gel. Various interactions intricately affect proteincrystallization. To enable gradual and slow changes in these variousinteractions, the present invention slowly brings the protein intocontact with the protein crystallization agents utilizing the diffusionphenomenon of the gel, thereby creating a state where the interactionbetween them and other interactions change gradually. As a result, bythe method of crystallizing protein, protein crystals with good qualitycan be precipitated conveniently and highly efficiently.

Furthermore, the present invention relates to a method of screening forprotein crystallization condition utilizing a microarray for proteincrystallization or a device for protein crystallization, which comprisesprotein crystallization agents held therein. Protein crystallization isaffected by parameters such as the purity and the concentration of theprotein, the type and the concentration of a precipitating agent, thetype and the concentration of a buffering agent or salt, pH, andtemperature. The crystallization conditions for a specific protein aredetermined by a combination of these parameters. The present inventionconveniently and quickly determines the optimum conditions from manyassumed crystallization conditions utilizing the microarray whereinthese parameters are highly integrated and using a small quantity of aprotein sample.

1. Method of Crystallizing Protein

The method of crystallizing protein comprises applying aprotein-containing sample to polymer gel holding protein crystallizationagents, and bringing the protein into contact with the proteincrystallization agents in the gel. Specifically, the method ofcrystallizing protein comprises the steps of (a) applying aprotein-containing sample to polymer gel holding protein crystallizationagents, and (b) bringing the protein into contact with the proteincrystallization agents.

Step (a): Applying to Polymer Gel

In step (a) of the method of crystallizing protein, a protein-containingsample is applied to polymer gel holding protein crystallization agents.Here, the protein-containing sample may further contain a proteinsolubilizing agent that help protein be dissolved, a stabilizing agentsuch as a reducing agent, or the like, in addition to protein to becrystallized. As the protein solubilizing agent, for example, asurfactant or the like that dissolves membrane protein can beexemplified. Furthermore, the polymer gel holding the proteincrystallization agents can be prepared as described below.

Types of polymer gel that can be used in the present invention are notspecifically limited. For example, gel that can be used herein isprepared by conducting, in an aqueous medium, for example,co-polymerization using 1, 2, or more types of monomers such asacrylamide, N,N-dimethylacrylamide, N-isopropylacrylamide,N-acryloylaminoethoxyethanol, N-acryloylaminopropanol,N-methylolacrylamide, N-vinylpyrrolidone, hydroxyethylmethacrylate,(meta)acrylic acid, or allyl dextrin, and multifunctional monomers, suchas methylenebis(meta)acrylamide, polyethylene glycol di(meta)acrylate,or the like. Additional examples of polymer gel that can be used in thepresent invention include gel such as agarose, alginic acid, dextran,polyvinyl alcohol, and polyethylene glycol, or gel prepared bycross-linking these.

To fill any container (e.g., microarray) with gel, a liquid containingmonomers such as acrylamide, multifunctional monomers, and an initiator,which are gel components, is poured into the chamber for polymerizationand gelatinization to proceed. Gelatinization may also be conducted byconducting co-polymerization in the absence of the multifunctionalmonomers and then using a cross-linking agent, in addition to a methodthat involves conducting co-polymerization in the presence of themultifunctional monomers. Moreover, in the case of agarose gel,gelatinization may be conducted by lowering temperature.

A method for causing the polymer gel to hold the protein crystallizationagents is not specifically limited. Here, “hold” means to immobilize theprotein crystallization agents in the polymer gel. For example, theprotein crystallization agents and the above polymerizable monomers maybe previously mixed and introduced into an appropriate chamber, so as tocause the formation of the polymer gel through the polymerizationprocess. Thus, the protein crystallization agents can be immobilized.Although the protein crystallization agents to be held by the polymergel differ depending on the type and the concentration of protein to becrystallized, persons skilled in the art can select appropriate proteincrystallization agents, appropriately set the concentrations and thequantities thereof, and thus can cause the polymer gel to hold theagents.

Alternatively, porous particles or the like may be impregnated with theprotein crystallization agents, and then the particles can be includedin the polymer gel.

For the protein crystallization agent, for example, conditions similarto those for commercially available Grid Screen™, Crystal Screen I & II,and Wizard™ I & II can be used.

To make it possible to observe over time how crystallization proceedsunder a microscope or the like, the polymer gel holding the proteincrystallization agents is preferably transparent. “Transparent” does notalways mean optical transmittancy of 100%. The polymer gel may betransparent to a degree such that how crystallization proceeds isobservable.

For example, when NaCl is held in gel, the gel is preferably preparedfrom acrylamide, 2-acrylamide-2-methylpropanesulfonic acid, ormethacryloyl oxyethyltrimethylammonium chloride. Furthermore, byappropriate selection of the above monomers and the like depending onthe types of the protein crystallization agents involved, the polymergel holding the transparent protein crystallization agents can beobtained.

To the above-prepared polymer gel holding the protein crystallizationagents, a protein-containing sample is applied. The protein-containingsample can be applied by any technique. For example, theprotein-containing sample may be applied by adding it dropwise to thepolymer gel holding the protein crystallization agents, filling thepolymer gel with the sample manually or mechanically using a syringe orthe like, or immersing the polymer gel in the protein-containing sample.The quantity of the protein-containing sample to be applied is setconsidering protein crystallization conditions and the like.

Step (b): Contact of Protein with Protein Crystallization Agent

As described above, after the protein-containing sample is applied tothe polymer gel holding the protein crystallization agents; the proteincomes into contact and reacts with the protein crystallization agents.In the method of crystallizing protein, the protein crystallizationagents gradually shift from within the gel to portions corresponding tothe protein-containing sample, or the protein-containing samplegradually shifts into the gel holding the protein crystallizationagents. Thus, the crystallization reaction occurs little by little.Accordingly, interaction between the protein and its surroundingenvironment (e.g., involving the presence of the protein crystallizationagents and pH levels), which is the crystallization mechanism, graduallychanges, so that crystals with good quality are precipitated.

After the application of the protein-containing sample, the polymer gelis allowed to stand in a state of being sealed or in the open air underappropriate temperature conditions for a time period sufficient for theprotein to be precipitated.

The time period sufficient for the protein to be precipitated differsdepending on a specific protein, concentration, crystallizationconditions, and the like, and ranges from approximately 1 hour to 10days. The appropriate temperature conditions also differ depending on aspecific protein, concentration, crystallization conditions, and thelike, and ranges from approximately 4° C. to 30° C.

After the time period sufficient for the protein to be precipitated haspassed, the state of precipitation of protein crystals is observed underan optical microscope, X-ray diffractometer, or the like. In the methodof crystallizing protein, a combination of known systems for monitoringprotein crystallization in the polymer gel can be used. For example, theway that crystals are precipitated can be recorded by taking picturesusing a CCD camera mounted in a microscope, and then the images areprocessed, so that whether crystallization is successful or not can bedetermined at a high speed.

As described above, the method of crystallizing protein comprisescausing a protein-containing sample with diffusion in the gel togradually come into contact with and react with protein crystallizationagents, thereby crystallizing the protein. Therefore, according to thepresent invention, with only a simple operation of applying aprotein-containing sample, protein crystals with good quality, which isappropriate for use in X-ray analysis or the like, can be obtained.Moreover, according to the method of crystallizing protein, as describedlater, for example, polymer gel holding the protein crystallizationagents is prepared on a microarray, and then the protein can becrystallized on the array. In this case, the quantities of the proteinto be used and the protein crystallization agent can be reduced. Evenwith the use of a small quantity of protein, good crystals can beobtained with high efficiency. Furthermore, the operation is convenient,so that the method is also appropriate for screening for proteincrystallization conditions.

2. Microarray for Protein Crystallization

The microarray for protein crystallization comprises at least 10different types and/or different concentrations of proteincrystallization agents held therein per 1 cm² of the microarray surface.

The microarray used in the present invention is not limited to amicroarray being made of specific material, a shape, or the like, aslong as it has a general configuration wherein many substances forreaction are arranged in order. Examples of materials used for themicroarray include glasses, resins, and polymer gel. In addition, acomplex prepared by combining these materials can also be used. For themicroarray of the present invention, materials with high opticaltransparency are preferably used in order to easily confirm proteinprecipitation.

In the microarray of the present invention, the shape of the array isnot specifically limited, as long as it meets requirements such thatupon the application of a protein-containing sample, the sample iseasily and uniformly dispensed to portions holding the proteincrystallization agents (hereinafter referred to as“protein-crystallization-agent-holding portions”), and that thisapportion can be performed quickly and efficiently without obstructingobservation of crystal precipitation. Examples of the shape of the arrayinclude a smooth shape having no extreme roughness, a shape havinggrooves formed thereon for apportioning a protein-containing sample tothe protein-crystallization-agent-holding portions, a shape having, forexample, circular or quadrangular reservoirs as theprotein-crystallization-agent-holding portions, and a shape using thesein combination.

To prevent a plurality of different types and different concentrationsof protein crystallization agents held in the microarray from mixingwith each other, a supplemental member for partition, which is made ofmaterial having no permeability for a solvent, is used integrated withthe microarray, so as to be able to create theprotein-crystallization-agent-holding portions. As such a supplementalmember, for example, members prepared by molding plate-shaped resinsinto a matrix shape, or non-porous hollow fibers prepared usingpolyethylene or the like can be used.

The number of the protein-crystallization-agent-holding portions ispreferably 10 or more, and more preferably 100 or more per 1 cm² of themicro array.

Furthermore, the microarray used in the present invention can have anyshape that does not obstruct observation of crystal precipitation, suchas circular, square, and rectangular shapes. The thickness of themicroarray can be appropriately selected considering improvement in theefficiency of crystallization and ease and speed of observation ofcrystal precipitation. For example, the microarray can have a thicknessbetween 0.1 and 5 mm, and preferably between 0.5 and 3 mm.

Next, as an example of the microarray used in the present invention, amicroarray comprising a plurality of tubulars arrayed therein isexplained in detail.

An example of the microarray comprising a plurality of tubulars arrayedtherein has a shape of a hollow-fiber-arrayed-thin-section 10 providedwith a plurality of hollow fibers 12 each having a hollow portion 11, asshown in FIG. 1. The hollow fibers used in the present invention arepreferably non-porous, and examples of the hollow fibers include thoseformed of monopolymers of methacrylate-based monomers such as methylmethacrylate, ethyl methacrylate, and butylmethacrylate; acrylate-basedmonomers such as methylacrylate and ethylacrylate; or copolymersthereof; polystyrene; polyethylene; norbornene/ethylene copolymers;polyethylene terephthalate; polycarbonate; or glass.

To obtain the thin sections of the hollow-fiber-array as shown in FIG.1, fibers are arrayed in parallel at certain intervals. In addition, toincrease the number of the protein-crystallization-agent-holdingportions per unit area, the external diameter of the hollow fiber ispreferably 1 mm or less, and more preferably 0.5 mm or less.Furthermore, in the microarray of the present invention, in view ofcausing the hollow portions to hold the protein crystallization agents,the internal diameter of the hollow fibers is preferably 10 μm or more,and more preferably 50 μm or more.

The hollow fibers may be used with their inner wall surfaces untreated,or may be used with their inner wall surfaces treated with plasma orradiation such as y-rays and electron beams, if necessary. Furthermore,a reactive functional group may be introduced into the hollow fibers, ifnecessary.

The hollow fibers prepared as described above can form a basic unit tocompose the microarray of the present invention comprising a pluralityof the hollow fibers arrayed therein. Then, these hollow fibers arebundled, and then adhered, thereby forming a fiber-array(three-dimensional-array).

The above three-dimensional-array is cut in a direction so as to crosswith the fiber axis, and is preferably cut in a direction vertically tothe fiber axis using a device for preparing sections, such as amicrotome, so that a thin section having cross sections of thehollow-fiber-arrayes can be obtained (FIG. 1). As for the thickness ofthe thin section, the thickness is generally between 100 and 5000 μm,and preferably between 500 and 3000 μm.

At this time, the hollow fibers are arrayed regularly, and then made toadhere using a resin adhesive or the like, so that, for example, ahollow-fiber-array wherein the hollow fibers are regularly arrayed inorder vertically and horizontally can be obtained. The shape of thehollow-fiber-array is not specifically limited, and is generally formedinto, for example, a square, rectangular, circular, or helical shape byregularly arraying the fibers.

“Regularly (array)” refers to an array chip in order so that the samenumber of the fibers is always contained in a frame with a certain size.For example, when fibers having a diameter of 1 mm are bundled andarrayed so as to form a square cross section 10 mm in length and 10 mmin width, 10 fibers are contained within one side of the square frame (1cm²). After these 10 fibers are bundled and placed in a row so as toform one layer of sheet, these sheets are stacked to form 10 layers ofsheet. As a result, 10 fibers can be arrayed lengthwise and 10 fiberscan be arrayed breadthwise, that is, 100 fibers can be arrayed in total.However, the technique for arraying fibers regularly is not limited tothe above wherein sheets are multilayered.

The number of fibers to be bundled in the present invention is 10 fibersor more, and preferably 100 fibers or more, and the number thereof canbe appropriately set depending on purposes.

Regarding details about the above-described microarray comprising aplurality of hollow fibers arrayed therein, see the WO 00/53736pamphlet.

The thin section of the hollow-fiber-arrays prepared as described abovehas a plurality of the protein-crystallization-agent-holding portions,and the protein crystallization agents held therein are not mixed, sothat the this section is preferred as the microarray of the presentinvention for protein crystallization.

The microarray of the present invention for protein crystallizationholds the protein crystallization agents directly or by means of gel orthe like. The “protein crystallization agent” means a compound that actsto lower the solubility of protein as described above, and examples ofthe agent include a precipitating agent, a pH buffering agent, and otheradditives.

As the precipitating agent, salt (e.g., sodium chloride, ammoniumsulfate, magnesium chloride, or calcium chloride), polyethylene glycolwith a molecular weight between 400 and 20000, and organic solvents(e.g., 2-methyl-2,4-pentanediol, ethanol, methanol, isopropanol, anddioxane) can be used alone, or a plurality of them can be used incombination.

As the pH buffering agent, sodium acetate trihydrate, potassiumphosphate, imidazole, sodium citrate, sodium cacodylate, and the likecan be used alone, or a plurality of these components can be mixed andused.

As the additives, monovalent salt, divalent salt, glycerol, surfactant,and the like can be used.

Moreover, as the protein crystallization agent, those based on knowndocuments and those prepared based on propositions made by users may beused.

In the present invention, when a plurality of protein crystallizationagents is used, the number thereof is appropriately determined dependingon different parameters, that is, a combination of concentrations andtypes of protein crystallization agents.

The present invention enables easy and quick screening for appropriateprotein crystallization conditions from a large number of pre-setprotein crystallization conditions by applying a large number of proteincrystallization agents set according to such different parameters to thesame experimental system (microarray).

In the microarray of the present invention, 10 or moreprotein-crystallization-agent-holding portions (e.g., hollow portions ofhollow fibers) are arrayed. Thus, 10 or more concentrations and types,and approximately a maximum of 1000 concentrations and types, can becombined. Accordingly, protein crystallization conditions can bescreened for exhaustively.

The concentrations of protein crystallization agents to be held in themicroarray of the present invention depend on types of the proteincrystallization agents, for example, in the case of polyethylene glycolas a precipitating agent, 5 to 50% by volume, and preferably 10 to 35%by volume; and in the case of a pH buffering agent, 0.05 to 0.5 mol/L,and preferably 0.1 to 0.2 mol/L. An object of the present invention isto cause protein crystallization agents at a plurality of concentrationsto be held on a microarray, and the protein crystallization agents atmulti-levels of concentrations within the above range are preferablyused. Specifically, for example, when sodium chloride is used as aprecipitating agent, on the same microarray, for example, 5-grade,10-grade, or 20-grade dilution series, or the like within a rangebetween 0.5 and 4.0 mol/L are prepared. Regarding the concentrations ofprotein crystallization agents to be held, the concentration ranges andthe concentration grades, or combinations thereof, can be appropriatelydetermined by persons skilled in the art considering types of theprotein crystallization agents and the like.

The quantities of protein crystallization agents to be held on themicroarray are not specifically limited, and can be appropriatelydetermined.

The above-described protein crystallization agents with a plurality oftypes and concentrations are held on the microarray. The term “hold”means to immobilize the protein crystallization agents on themicroarray.

A method for causing protein crystallization agents to be held in theprotein-crystallization-agent-holding portions in the microarray, thatis, reservoirs such as grooves or the hollow portions of the hollowfibers, is not specifically limited. For example, proteincrystallization agents may be directly held in theprotein-crystallization-agent-holding portions in the microarray, orheld by means of polymer gel or the like. Moreover, the proteincrystallization agents impregnated in porous particles or the like canbe included in polymer gel. Furthermore, a method that can also beemployed herein involves mixing the protein crystallization agents withpolymerizable monomers and previously introducing the resultant into theprotein-crystallization-agent-holding portions of the microarray, andthen forming polymer gel through a polymerization process, so as toconduct immobilization.

The types of gel that can be used in the present invention are notspecifically limited. For example, gel that can be used herein can beprepared by copolymerization using 1, 2, or more types of monomers suchas acrylamide, N,N-dimethylacrylamide, N-isopropylacrylamide,N-acryloylaminoethoxyethanol, N-acryloylaminopropanol,N-methylolacrylamide, N-vinylpyrrolidone, hydroxyethylmethacrylate,(meta)acrylic acid, or allyldextrin, and multifunctional monomers, suchas methylenebis(meta)acrylamide, polyethyleneglycoldi (meta)acrylate, orthe like, for example, in an aqueous medium. Further examples of gelthat can be used in the present invention include gel such as agarose,alginic acid, dextran, polyvinyl alcohol, and polyethylene glycol, orgel prepared by crosslinking the same.

To fill the protein-crystallization-agent-holding portions of themicroarray with gel, a liquid containing monomers such as acrylamide,multifunctional monomers, and an initiator, which are gel components,are injected into the protein-crystallization-agent-holding portions ofthe microarray, followed by polymerization and gelatinization. Inaddition to a method that involves conducting co-polymerization in thepresence of multifunctional monomers, gelatinization may also beconducted using a cross-linker after co-polymerization in the absence ofmultifunctional monomers. Furthermore, in the case of agarose gel,gelatinization may be conducted by lowering temperature.

As described above, the microarray of the present invention for proteincrystallization can be obtained. The prepared microarray is preferablystored in a container that is sealed well so as to prevent contact withthe open air. Examples of material for a container for storage includepolymer materials having a small degree of gas or water permeability, orglass and metal. The microarray is preferably stored at a lowtemperature. When the microarray is stored for particularly a long timeperiod, it can also be freezed.

3. Device for Protein Crystallization

The device for protein crystallization is provided with (a) a microarrayfor protein crystallization havingprotein-crystallization-agent-holding-portions, which comprises at least10 different types and/or different concentrations of proteincrystallization agents held therein per 1 cm² of the microarray surface;and (b) a plate, which has wells that can be filled with aprotein-containing sample, corresponding to eachprotein-crystallization-agent-holding-portion of the microarray (a)above for protein crystallization.

Configuration of such a device enables the achievement of furthersmaller quantities of required protein samples, simplification ofexperimental protocols for crystallization, and smaller space requiredfor experiments by an ultrahigh degree of integration. Moreover, it canease quantification of a protein-containing sample and observation of astate of crystallization under a microscope.

(a) Microarray for Protein Crystallization

In the device for protein crystallization, as a member (a), themicroarray for protein crystallization described in the above section“2. Microarray for protein crystallization” can be used.

Furthermore, the microarray (a) for protein crystallization may be fixedonto a support such as a flat plate. The support that can be used hereinis not specifically limited, as long as it has a shape and is made ofmaterial to which the microarray can be fixed. For example, as thematerial for the support, glass, resin, and metal can be exemplified.The material for the support can be appropriately selected from them,and one type alone or 2 types or more of material can be used incombination. The support to which the microarray for proteincrystallization is fixed is preferably prepared from, in particular, anoptically transparent material (e.g., slide glass). By the use of theoptically transparent material, the thus generated crystals can bequickly and conveniently confirmed under a microscope while keeping thecrystals in the device, and the growth process of the crystals can beobserved over time under a microscope.

Furthermore, it is preferred to make a marking on the above support soas to determine a position to which the microarray for proteincrystallization is fixed. A method for marking is not specificallylimited, as long as the microarray for protein crystallization can befixed precisely. An appropriate method involves forming a frame patternhaving an external shape along the circumference of the microarray whileadhering it to the support. In this case, the material for the framepattern for marking is not specifically limited, and stainless steel isappropriate.

(b) Plate

In the device for protein crystallization, the member (b) is a platehaving wells that can be filled with a protein-containing sample,corresponding to each protein-crystallization-agent-holding portion ofthe microarray (a) above for protein crystallization. This plate istypically shown in FIG. 2. In FIG. 2, wells 21 that can be filled with aprotein-containing sample are formed on a plate 20. However, the plateconfiguration shown in this figure is an example, and the configurationis not limited thereto.

The plate that can be used in the present invention is not specificallylimited, as long as it has a shape and is made of material so that itcan be superposed on the microarray (a) for protein crystallization, andcan be selected or designed appropriately in consideration of the shapeof the microarray (e.g., with a smooth shape without any roughness). Asthe material for the plate, glass, resin, metal, and the like can beexemplified. The material for the plate can be appropriately selectedfrom them, and one type alone or 2 types or more of material can be usedin combination. To observe a state of protein crystallization, the platehas preferably transparent (optically transparent) portions that aresuperposed on the microarray (a) for protein crystallization,corresponding to each protein-crystallization-agent-holding portion ofthe array chip. Hence, the plate is particularly preferably made of anoptically transparent material (e.g., transparent resin or glass).

On the plate (b), the wells that can be filled with a protein-containingsample are formed. The plate has at least 10 wells per 1 cm² of theplate, corresponding to the protein-crystallization agent-holdingportions of the microarray (a) for protein crystallization. The methodfor forming the wells is not specifically limited. For example, at least10 holes may be regularly created per 1 cm² of any plate material, andthen the plate material may be adhered to a substrate, thereby formingthe wells. The plate material is not limited, and can be made ofmaterial such as metal, resin, and rubber. In particular, the platematerial is preferably made of stainless steel. As a technique to createholes on the plate material, a technique such as injection molding,drilling, laser processing, optical molding, or etching can be employed.The substrate to which the plate material is adhered is preferablytransparent (optically transparent), because the substrate portion issuperposed in a manner corresponding to eachprotein-crystallization-agent-holding portion of the array chip. Hence,the substrate is preferably made of an optically transparent material(e.g., slide glass).

The shape of the well to be formed is not specifically limited, as longas the wells can be arranged so as to be superposed in a mannercorresponding to each protein-crystallization-agent-holding portion ofthe microarray, and can be filled with a protein-containing sample. Forexample, in terms of ease of formation of the well, the wells arepreferably lattice-shaped. The capacity of the well is not specificallylimited, as long as it is sufficient for filling the well with aprotein-containing sample. When reduction of protein quantity, which isone of the purposes of the present invention, is taken intoconsideration, the capacity of the well is preferably 1 μL or less, morepreferably 100 nL or less, and further preferably 50 nL or less.

In the device for protein crystallization, the above device (a) forprotein crystallization is used with the plate (b) superposed thereon.FIG. 3 typically shows how the microarray for protein crystallizationand the plate are arranged on the device for protein crystallization. InFIG. 3, a microarray 31 for protein crystallization is fixed on asupport 33 on which a marking 34 is made to determine the position forthis fixation. In the device for protein crystallization, a spacer 35 ispreferably placed between the support 33 and the plate 32, which has aspace into which the microarray 31 for protein crystallization fixed onthe support 33 can be accommodated. The spacer 35 is a supplementalmember for fixing the microarray 31 fixed on the support 33 at a fixedposition, and by which it becomes possible to place each well part ofthe plate 32 in a manner corresponding to each hollow portion of themicroarray 31.

The shape and the material for the spacer are not specifically limited,as long as the spacer is appropriate for use in combination with othercomponents of the device for protein crystallization (e.g., themicroarray for protein crystallization, the plate, or the support).Examples of material that can be appropriately used include metal,resin, and rubber. The spacer 35 is preferably made of stainless steelin terms of ease for the formation of the space, strength, salttolerance, and the like.

The size of the space is not particularly limited as long as it issufficient for the microarray 31 for protein crystallization to beaccommodated. A particularly preferred size enables the microarray 31for protein crystallization to be accommodated without any gap.Furthermore, the thickness of the spacer preferably enables themicroarray 31 for protein crystallization fixed on the support 33 to bein contact with the plate 32 without any gap.

The device for protein crystallization comprises a combination of themicroarray for protein crystallization having various proteincrystallization conditions integrated on a sheet of the array chip, andthe plate having wells that can be filled with a protein-containingsample. Hence, the plate is filled with a protein-containing sample, andthen the plate is superposed on the microarray for proteincrystallization, so that the protein-crystallization-agent-holdingportions on the array chip and the wells filled with theprotein-containing sample are superposed corresponding to each other,thereby bringing the protein into contact with and to react with theprotein crystallization agents. Here, a technique for filling the wellswith a protein-containing sample can be a known technique, such as atechnique that involves simply pouring the protein-containing sampleinto the wells. In addition, the order of superposing (up or down) themicroarray for protein crystallization and the plate is not specificallylimited.

In the device for protein crystallization, partitions for eachprotein-crystallization-agent-holding portion are specified by the useof the plate, so as to be able to prevent samples or other substancesfrom shifting or mixing (contamination) among arrays, when aprotein-containing sample is applied to the arrays.

4. Screening for Crystallization Conditions

The method of the present invention for screening for proteincrystallization conditions comprises, using the above microarray forprotein crystallization or the above device for protein crystallization,a step of precipitating protein by bringing protein crystallizationagents into contact with a protein-containing sample on and/or in themicroarray.

In the present invention, a protein-containing sample is a samplecontaining protein whose crystallization conditions are to be specified.Examples of the protein include natural or synthetic peptides,polypeptides, protein, and protein complexes. These substances arepreferably extracted or isolated, or generated by a genetic engineeringtechnique, a chemical synthesis technique, or the like from natural orsynthetic sources, and then previously purifying protein to be screenedfor by a combined use of general purification methods such as solventextraction, column chromatography, and liquid chromatography.

Since protein concentration and purity in a protein-containing sampleare also factors in protein crystallization conditions,protein-containing samples may be prepared at several grades ofconcentration and purity, and then allowed to react with proteincrystallization agents. For example, protein concentration is preferablychanged at several grades within a range between 5 and 30 mg/ml. Inaddition, the quantity of a protein-containing sample for the reactionwith protein crystallization agents can be appropriately changeddepending on array size.

The protein-containing sample may contain, in addition to protein to bescreened for, a protein solubilizing agent to help protein be dissolved,a protein stabilizing agent such as a reducing agent, or the like. Asthe protein solubilizing agent, for example, a surfactant to dissolve,for example, membrane protein can be exemplified.

The method of the present invention of screening for proteincrystallization conditions comprises the step of precipitating proteinby bringing the above protein-containing sample into contact with theprotein crystallization agents in the above microarray or the abovedevice. Here, “bringing into contact” refers, for example, to add aprotein-containing sample dropwise to theprotein-crystallization-agent-holding portions, filling the portionswith the sample manually or mechanically using a syringe or the like, orimmersing the microarray in the protein-containing sample.

After the protein crystallization agents are allowed to react with theprotein-containing sample in the microarray or the device, themicroarray or the device is allowed to stand in a state of being sealedor in the open air under a specific temperature condition for a timeperiod sufficient for protein to be precipitated.

The time period that is sufficient for protein to be precipitateddiffers depending on a specific protein, concentration, crystallizationconditions, and the like, and ranges from approximately 1 hour to 10days. When no crystals are precipitated even after 30 days or more havepassed, the crystallization conditions therefor are determined to beinappropriate.

Since the temperature condition is a factor in protein crystallizationconditions, the screening method may be conducted by changing thetemperature condition. The temperature condition is preferably set atseveral grades such as at 4° C., 15° C., 18° C., or 22° C.

After time period sufficient for protein to be precipitated has passed,the way that protein crystals are precipitated is observed using, forexample, an optical microscope or an X-ray diffractometer.

When protein crystallization conditions are screened for using themicroarray or the device of the present invention, known systems formonitoring protein crystallization in the microarray or the device canbe used in combination. Specifically, the way that crystals areprecipitated in many crystallization conditions in the microarray or thedevice can be recorded by, for example, taking pictures using a CCDcamera mounted in a microscope, and then processing the images, so thatwhether crystallization is successful or not can be determined at a highspeed.

When protein crystals are confirmed, the confirmed conditions aredetermined as crystallization conditions for the protein. Alternatively,to narrow down conditions to more detailed protein crystallizationconditions, a second-stage screening method can be conducted whereinprotein crystallization conditions are set within a narrower range.

When protein crystallization conditions are screened for in this manner,using the crystallization conditions, protein is precipitated from theoriginal protein sample by the above-described method for crystallizingprotein or a method for crystallizing protein that is generally employedin the art such as a hanging drop method, a sitting drop method, adialysis method, or a batch method.

The microarray or the device for protein crystallization of the presentinvention comprises a plurality of protein crystallization conditionsintegrated therein. The use of the microarray or the device of thepresent invention enables screening for appropriate conditionssimultaneously from a plurality of protein crystallization conditionsusing a small quantity of protein, and thus is useful in easily andquickly determining protein crystallization conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an oblique view typically showing a hollow-fiber-arrayed thinsection containing gel, which is an example of a microarray for proteincrystallization according to the present invention.

FIG. 2 is an oblique view typically showing a plate that is superposedon a microarray for protein crystallization in a device for proteincrystallization.

FIG. 3 is an oblique view typically showing arrangement of themicroarray for protein crystallization and the plate in the device forprotein crystallization.

FIG. 4 is a microphotograph showing a lysozyme in a crystal state usingsodium chloride as a crystallization agent on the hollow fiber-arrayedthin section containing gel.

Explanation of Symbols

-   10 Hollow-fiber-arrayed thin section-   11 Hollow portion-   12 Hollow fiber-   20 Plate-   21 Well-   31 Microarray for protein crystallization-   32 Plate-   33 Support-   34 Marking-   35 Spacer

BEST MODE OF CARRYING OUT THE INVENTION

The present invention will be described in more detail with reference tothe following examples, but the present invention is not limited tothese examples.

EXAMPLE 1 Preparation of Hollow-Fiber-Array

A porous plate has holes which have a diameter of 0.32 mm, and a centerdistance of which holes is 0.42 mm. Specifically, 10 lines were providedlengthwise and breadthwise, respectively, that is, 100 holes are arrayedin total. A sheet of the porous plate with a thickness of 0.1 mm wassuperposed on another sheet of the porous plate. Through each hole ofthe two sheets of the porous plates, 100 hollow fibers made ofpolyethylene (with an external diameter of approximately 300 μm,internal diameter of approximately 160 μm, and length of approximately50 cm) (MITSUBISHI RAYON CO., LTD.) were passed. The distance betweenthe 2 sheets of the porous plates was determined to be 30 cm. The hollowfibers were kept strained and fixed at two positions: a position 10 cmaway from and a position 40 cm away from one end of the hollow fibers.

Next, resin materials were poured between 2 sheets of the porous plates.As the resin, a polyurethane resin adhesive (NIPPON POLYURETHAN INDUSTRYCO., LTD., NIPPOLLAN 4276 and CORONATE 4403) was used after addingcarbon black, which was 2.5% by mass of the total weight of theadhesive, to the adhesive. The resins were hardened by allowing theresultant to stand at room temperature for 1 week. Next, the porousplates were removed, so that a hollow-fiber-array was obtained.

EXAMPLE 2 Introduction and Immobilization of Polymer Gel toHollow-Fiber-Array

A mixed solution consisting of the following compositions was prepared.Acrylamide 3.7 parts by mass Methylene bisacrylamide 0.3 parts by mass2,2′-azobis (2-amidinopropane) dihydrochloride 0.1 parts by mass

The mixed solution and the hollow-fiber-array obtained in Example 1 wereplaced in a desiccator. The non-arrayed end parts (free end parts) ofthe hollow-fiber-arrays were immersed in the mixed solution, and thenthe desiccator was reduced to pressure to introduce the mixed solutionwas introduced into the hollow portions of the hollow fibers.Subsequently, the hollow-fiber-array was transferred to a sealed glasscontainer having an interior saturated with water vapor, followed by apolymerization reaction at 80° C. for 4 hours. Thus, ahollow-fiber-array wherein gelatinized products were immobilized in thehollow portions was obtained. As a result, a hollow-fiber-array holdingacrylamide gel inside was obtained.

EXAMPLE 3 Preparation of Hollow-Fiber-Array Thin Section Containing Gel

The hollow-fiber-array containing acrylamide gel obtained in Example 2was sliced in a direction perpendicular to the fiber axis to have athickness of approximately 2 mm using a micrtome, thereby obtaining ahollow-fiber-array thin section on which 10-by-10 (length-to-width),that is, 100 bundles in total, of the hollow fibers containing gel wereregularly arrayed to form a square (FIG. 1). FIG. 1 shows thehollow-fiber-array thin section containing gel as prepared throughExamples 1, 2, and 3. The hollow portions 11 of the hollow fibers 12were filled with the gel prepared in Example 2.

EXAMPLE 4 Example of Screening for Crystallization Conditions UtilizingHollow-Fiber-Array Thin Section Containing Gel, and ProteinCrystallization in Gel

The hollow-fiber-array thin section containing gel (the microarray)obtained in Example 3 was freeze-dried. One bundle of the hollow fibersof the microarray was selected, to which 1 μL of a 2 mol/L sodiumchloride aqueous solution was added dropwise as a proteincrystallization agent (spot A). Subsequently, to other bundles of thehollow fibers on the same microarray, crystallization agents shown belowwere each added dropwise (spots B, C, D, E, F, G, H, I, and J) in amanner similar to the above procedure. The resultants were allowed tostand for several minutes. The protein crystallization agents were heldby infiltrating these solutions into the gel.

Crystallization Agents

-   Spot A: 2.0 mol/L sodium chloride aqueous solution-   Spot B: 0.5 mol/L sodium chloride aqueous solution-   Spot C: 10% by volume polyethylene glycol (molecular weight of 400)    aqueous solution-   Spot D: 20% by volume polyethylene glycol (molecular weight of 400)    aqueous solution-   Spot E: 10% by volume polyethylene glycol (molecular weight of 6000)    aqueous solution-   Spot F: 20% by volume polyethylene glycol (molecular weight of 6000)    aqueous solution-   Spot G: 20% by volume 2-methyl 2,4-pentanediol aqueous solution-   Spot H: 40% by volume 2-methyl 2,4-pentanediol aqueous solution-   Spot I: 0.5 mol/L ammonium sulfate aqueous solution-   Spot J: 1.5 mol/L ammonium sulfate aqueous solution

10 μL of an aqueous solution (80 mg/ml) of lysozyme (manufactured bySigma-Aldrich Corporation) which is an enzyme that degradespolysaccharides, was spotted on the microarray using a 20 μL autopipette, and then dispersed uniformly on the microarray. The resultantwas allowed to stand at 20° C. for 3 hours. As a result of observationunder an optical microscope, no crystals of the lysozyme were observedat the spot B that had been infiltrated with the polyethylene glycolaqueous solution, and columnar crystals that are optimum for X-raystructural analysis were observed at the spot A that had beeninfiltrated with the sodium chloride aqueous solution (2 mol/L). (FIG.4).

Next, based on the above results, approximately 0.3 mm×0.3 mm×0.5 mmlysozyme crystals were obtained by the hanging drop vapor diffusionmethod from the lysozyme-containing sample solution using 2 mol/L sodiumchloride as a precipitating agent.

According to the above results, screening was performed using sodiumchloride, polyethylene glycol, 2-methyl-2,4-pentanediol, and ammoniumsulfate, revealing that sodium chloride at a concentration of 2 mol/L isa condition appropriate for protein crystallization.

Furthermore, as shown in FIG. 4, by crystallizing protein in the gelholding the protein crystallization agents according to the presentinvention, columnar crystals that are optimum for X-ray structuralanalysis were obtained. Hence, it was shown that the method ofcrystallizing protein of the present invention is a convenient anduseful method of crystallizing protein and screening therefor.

INDUSTRIAL APPLICABILITY

According to the present invention, a method of precipitating proteincrystals from a protein-containing sample, a novel microarray, and anovel device for screening for conditions for precipitating proteincrystals are provided. By the present protein crystallization method,crystals with good quality can be obtained conveniently and highlyefficiently. Moreover, by the use of the microarray or the device havingcrystallization conditions highly integrated therein, proteincrystallization conditions can be screened for conveniently and quicklyeven with an extremely small quantity of a sample.

1. A method of crystallizing protein, which comprises the followingsteps of: (a) applying a protein-containing sample to polymer gelholding protein crystallization agents; and (b) bringing the proteininto contact with the protein crystallization agents.
 2. The method ofcrystallizing protein of claim 1, wherein the protein-containing samplecontains protein, a protein solubilizing agent, and/or an additive. 3.The method of crystallizing protein of claim 1, wherein the proteincrystallization agent contains a precipitating agent and/or a pHbuffering agent.
 4. The method of crystallizing protein of claim 1,wherein the polymer gel holding the protein crystallization agents istransparent.
 5. A microarray for protein crystallization, whichcomprises at least 10 different types and/or different concentrations ofprotein crystallization agents held thereon per 1 cm² of the microarraysurface.
 6. The microarray of claim 5, wherein the proteincrystallization agent contain a precipitating agent and/or a pHbuffering agent.
 7. The microarray of claim 5 or 6, wherein the proteincrystallization agents are held by means of the polymer gel.
 8. Themicroarray of claim 7, wherein the polymer gel holding the proteincrystallization agents is transparent.
 9. The microarray of claim 5,which comprises a plurality of hollow fibers arrayed therein holdingdifferent types and/or different concentrations of the proteincrystallization agents in the hollow portions thereof.
 10. Themicroarray of claim 9, wherein the protein crystallization agents areheld by means of the polymer gel that fills the hollow portions.
 11. Themicroarray of claim 9 or 10, wherein the protein crystallization agentcontains a precipitating agent and/or a pH buffering agent.
 12. A devicefor protein crystallization, which is provided with the following (a)and (b): (a) a microarray for protein crystallization havingprotein-crystallization-agent-holding portions that comprise at least 10different types and/or different concentrations of proteincrystallization agents held thereon per 1 cm² of the microarray surface;and (b) a plate having wells that can be filled with aprotein-containing sample, and correspond to eachprotein-crystallization-agent-holding portion of the microarray (a) forprotein crystallization.
 13. The device for protein crystallization ofclaim 12, wherein in (a), the protein crystallization agents are held bymeans of polymer gel.
 14. The device for protein crystallization ofclaim 12 or 13, wherein in (a), the protein crystallization agentcontains a precipitating agent and/or a pH buffering agent.
 15. Thedevice for protein crystallization of claim 12, wherein in (a), themicroarray for protein crystallization comprises a plurality of hollowfibers arrayed thereon holding different types and/or differentconcentrations of the protein crystallization agents in the hollowportions thereof.
 16. The device for protein crystallization of claim12, wherein in (b), the capacity of the well is 1 μL or less.
 17. Thedevice for protein crystallization of claim 12, wherein in (b), whensuperposed on the microarray (a) for protein crystallization, thesuperposed portion corresponding to eachprotein-crystallization-agent-holding portion of the array istransparent.
 18. The device for protein crystallization of claim 12,wherein in (b), the plate is prepared from an optically transparentmaterial.
 19. The device for protein crystallization of claim 18,wherein the plate is made of a transparent resin or glass.
 20. Thedevice for protein crystallization of claim 12, wherein in (b), theplate has a structure in which a plate material perforated regularly isadhered to a substrate.
 21. The device for protein crystallization ofclaim 20, wherein the substrate is made of an optically transparentmaterial.
 22. The device for protein crystallization of claim 20,wherein the substrate is prepared from a slide glass.
 23. The device forprotein crystallization of claim 20, wherein the plate material is madeof material selected from the group consisting of metal, resin, andrubber.
 24. The device for protein crystallization of claim 12, whereinthe microarray (a) for protein crystallization is fixed on a support.25. The device for protein crystallization of claim 24, wherein thesupport is made of an optically transparent material.
 26. The device forprotein crystallization of claim 24, wherein the support is preparedfrom a slide glass.
 27. The device for protein crystallization of claim24, wherein marking is made on the support so as to determine a positionfor fixing the microarray (a) for protein crystallization.
 28. Thedevice for protein crystallization of claim 24, wherein a spacer isplaced between the support and the plate (b), the spacer having a spaceinto which the microarray (a) for protein crystallization fixed on thesupport can be accommodated.
 29. The device for protein crystallizationof claim 28, wherein the spacer is made of material selected from thegroup consisting of metal, resin, and rubber.
 30. A method of screeningfor protein crystallization conditions, which comprises a step ofprecipitating protein by bringing the protein crystallization agentsinto contact with a protein-containing sample on and/or in themicroarray, using the microarray claim 5, or the device of claim
 12. 31.The method of claim 30, wherein the protein-containing sample containsprotein, a protein solubilizing agent, and/or an additive.
 32. Themethod of claim 30, which is conducted under a plurality of temperatureconditions.
 33. The method of claim 30, wherein the volume of theprotein-containing sample solution is 1 μL or less per type orconcentration of the protein crystallization agent.
 34. A method ofscreening for protein crystallization conditions, which comprises a stepof precipitating protein by superposing the wells of (b) filled with theprotein-containing sample onto the protein-crystallization-agent-holdingportions holding the protein crystallization agents on the microarray(a), so as to bring the protein crystallization agents into contact withthe protein in the device for protein crystallization of claim 12.